The Transforming Growth Factor-beta (TGF-.beta.) family consists of many structurally related small peptides, which regulate a wide range of crucial cell growth and differentiation events, including early embryonic patterning and morphogenesis, sexual organ and bone/cartilage formation, wound healing and immunosuppression. Disregulation of these events has been implicated in a variety of diseases including tumorigenesis (Massague, J. et al., Trends in Cell Biology 4:172-178 (1994); Kingsley, D. M., Genes Dev. 8:133-146 (1994); Roberts, A. B. and Sporn, M. B., Growth Factors 8:1-9 (1993); Attisano, L. et al., Biochim. Biophys. Acta. 1222:71-80 (1994); Moses, H. L. et al., Cell 63:245-247 (1990); Border, W. A. and Ruoslahti, E. J., J Clin. Invest. 90:1-7 (1992)). However, the molecular mechanisms involved in these processes are not fully defined.
Recent progress in the cloning and characterization of the cell surface receptors for each member of the family yielded two new groups of receptors, the type I and type II, which consist of homologous single transmembrane serine/threonine kinases (for reviews, see Massague, J. et al., Trends in Cell Biology 4:172-178 (1994); Derynck, R., TIBS:548-553 (1994); Mathews, L. S., Endocr. Rev. 15:310-324 (1994); Heldin, C.-H., Cell 80:213-223 (1995)). Unlike most of the growth stimulatory factors, which mediate their cellular effects through homodimeric complexes of tyrosine kinase receptors (for reviews, see Schlessinger, J. and Ulrich, A., Neuron 9:383-391 (1992); Heldin, C.-H., Cell 80:213-223 (1995)), TGF-.beta. family members require heteromeric complexes of both type I and type II serine/threonine kinase receptors for signaling (reviewed by Massague, J. et al., Trends in Cell Biology 4:172-178 (1994); Heldin, C.-H., Cell 80:213-223 (1995)). Molecular characterization of the type I and the type II receptors of the prototypical TGF-.beta. revealed that the two types of receptors play different roles in mediating downstream signaling. Type II is a constitutively active serine/threonine kinase receptor which can bind TGF-.beta. independently, but cannot signal without the type I receptor (Laiho, M. et al., Cell 62:175-185 (1990); Wrana, J. L. et al., Cell 71:1003-1014 (1992)); while the type I receptor cannot bind to TGF-.beta. in the absence of the type II receptor, is inactive as a kinase on its own, but is likely activated by the type II receptor upon ligand binding (Wrana, J. L. et al., Nature 370:341-47 (1994); Ebner, R. et al., Science 260:1344-1348 (1993)). The recent discovery of a mutant type I receptor with a constitutively active kinase, able to signal in the absence of the type II receptor and TGF-.beta., suggests that the type I receptor is responsible for initiating the downstream signaling pathway subsequent to its activation by the type II receptor (Wieser, R. et al., EMBO J. 14:2199-2208 (1995)).
Little is known about how the type II receptor activates the type I receptor. Upon TGF-.beta. binding, the type II receptor forms heteromeric complexes with the type I receptor and phosphorylates the type I receptor (Wrana, J. L. et al., Nature 370:341-47 (1994)). Although all phosphorylation sites on the type I receptor have not yet been mapped, a serine/threonine rich domain located immediately amino-terminus to the kinase domain of the type I receptor (the GS box) was found to be phosphorylated (Wrana, J. L. et al., Nature 370:341-47 (1994); Wrana, J. L. et al., Mol. Cell. Biol. 14:944-950 (1994)). Type II receptor-mediated type I receptor phosphorylation can activate the type I receptor through two possible mechanisms: 1) by creating binding sites for activators or abolishing binding sites for inhibitors or, 2) by simply inducing a conformational change of the type I receptor, which could either directly activate the kinase activity or indirectly activate the kinase activity by releasing an inhibitor. To fully understand the molecular details involved in the activation of the type I receptor, it is therefore essential to identify and characterize the cytoplasmic interactors of the type I receptors before and after ligand binding.
The immunophilin FKBP12 has been reported as a specific cytoplasmic interactor of one member of the TGF-.beta. receptor family (Wang, T. W. et al., Science 265:674-676 (1994)). FKBP12 is known to mediate the immunosuppressive activities of two macrolides, FK506 and rapamycin, by binding to the macrolides and then recruiting and thereby inactivating the serine/threonine phosphatase calcineurin and the serine kinase FRAP (or RAFT1) respectively, resulting in the blockage of the signaling pathways mediated by calcineurin or FRAP (Siekierka, J. J. et al., Nature 341:755-777 (1989); Harding, M. W. et al., Nature 341:758-760 (1989); Bierer, B. E. et al., Proc. Natl. Acad. Sci. 87:9231-9235 (1990); Liu, J. et al., Cell 66:807-815 (1991); Liu, J. et al., Biochemistry 31:3896-3901 (1992); Clipstone, N. A. and Crabtree, G. R., Nature 357:695-697 (1992); O'Keefe, S. J. et al., Nature 357:692-694 (1992); Jain, J. et al., Nature 365:352-355 (1993); McCaffrey, P. G. et al., Chem. 268:3747-3752 (1993); Brown, E. J. et al., Nature 369:756-758 (1994); Sabatini, D. M. et al., Cell 78;35-43 (1994); Zheng, X.-F. et al., Cell 82:121-130 (1995); Brown, E. J. et al., Nature 377:441-446 (1995)). In the absence of the macrolides, however, FKBP12 cannot interact with either enzyme. Since high levels of FKBP12 are found in virtually all mammalian cell types and it is also highly conserved from plants to mammals, its physiological role is likely to be very important. Clearly, there is a need for further elucidation of the role FKBP12 plays in the TGF-.beta. receptor-mediated signaling pathway.